The main objective of this research is to analyze the processes of DNA repair and DNA replication in human fibroblasts grown from donors of different ages. This analysis will test whether the accuracy and activity of these processes decline as the donor age increases. The DNA repair and DNA replication properties of early passage cells grown from young and old donors will be compared to reveal differences which may be associated with in vivo aging. In addition, the properties of early passage cells grown from old donors will be compared to those of late passage cells from young donors in order to test whether DNA repair and DNA replication mechanisms undergo similar changes during in vivo and in vitro aging. Of particular interest is the activity of error-prone mechanisms which might lead to an accumulation of mutations in aging cells. The activity and error-prone character of DNA repair mechanisms as a function of age will be determined by making the following measurements: (1) cellular survival after ultraviolet (UV) light or DNA cross-link damage; (2) ability to repair UV-irradiated herpes virus (host cell reactivation); (3) cellular mutation frequencies after UV-irradiation; (4) enhancement of virus survival and mutagenesis on pre-irradiated host cells (inducible error-prone repair); (5) increased genetic recombination between infecting viral mutants damaged with UV light or DNA cross-links; and (6) extent of excision and post-replication repair of UV damage. The activity and accuracy of DNA replication in cells of different ages will be studied by the following procedures: (1) measurement of spontaneous cellular mutation frequencies; (2) measurements of the activity and fidelity of DNA polymerase gamma on both synthetic and natural polynucleotide templates; and (3) analysis of extracts of UV-treated cells for a possible inducible, error-prone DNA polymerase.